CN106754639A - A kind of mescenchymal stem cell factor large-scale producing method - Google Patents
A kind of mescenchymal stem cell factor large-scale producing method Download PDFInfo
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Abstract
The present invention relates to a kind of mescenchymal stem cell factor large-scale producing method, in vitro culture including mescenchymal stem cell, the secretion inducing of the mescenchymal stem cell factor and the mescenchymal stem cell factor isolate and purify three steps, and wherein the inducing culture needed for the secretion inducing step of the mescenchymal stem cell factor is 30 80 by volume ratio:20‑60:0.2 1.5 DMEM, the mixed culture medium of PBS and L alanyl L glutamine aqueous solutions and derivant composition.Mescenchymal stem cell factor large-scale producing method provided by the present invention, using single samples sources large-scale culture, ensure batch products homogeneity, the secretion inducing stage of the mescenchymal stem cell factor uses inducing culture, a large amount of secretions of stimulating cytokine, rationally effectively, whole preparation process conditional stability is rationally, suitable for mass production for formula.
Description
Technical field
The invention belongs to cell factor technical field, more particularly to a kind of mescenchymal stem cell factor side of preparation on a large scale
Method.
Background technology
Source for mesenchymal stem cells belongs to multipotential stem cell in the mesoderm and ectoderm of mesoderm growing early stage.Because its have it is many
To differentiation potential, hematopoiesis support and promote stem cell implantation, immunoregulation and be increasingly subject to people's the features such as self-replacation
Concern.
The mescenchymal stem cell mechanism of action is not completely clear and definite, it is considered that mainly by following mechanism：1st, paracrine is made
With secretory cell trophic factors, Porcine HGF, anti-apoptosis factor etc..2nd, instead of or repair dead or impaired cell.3、
Cell directly contact, the function of regulating cell.Currently, more and more research shows, the paracrine effect of mescenchymal stem cell
Its Main Function in it plays therapeutic effect.Mescenchymal stem cell produces work by secreting various types of cells factor pair adjacent cells
With so as to play its effect.Research shows that mescenchymal stem cell can secrete cytokine profiles, such as VEGF vascular endothelial growths
The factor, bFGF basic fibroblast growth factors, NGF nerve growth factors, HGF HGFs, IGF-1 insulin
Like growth factor 1, BDNF BDNFs, GDNF glial cell line-derived neurotrophic factors, IL-6 interleukin-6s,
IL-11 interleukin-11s etc..The mescenchymal stem cell secretion activity factor has improves inflammatory environment, regulation immunity of organism shape
State, suppression Apoptosis, promotion cell propagation, promotion revascularization, promotion NSC migrate and break up, promote aixs cylinder to give birth to
Long and Synaptic junction forms and promotes function that myelin is formed etc., by the animal model of Infant Injury in White Matter confirms that place can be suppressed
Chief cell and transplanted cells apoptosis, the nervous function for improving model mouse, confirm that heart function can be promoted by heart infarction animal model
Recover, testing confirmation by skin injury can promote wound healing.
It is public in the report of the preparation method of the current mescenchymal stem cell factor, such as Chinese patent application CN105543313A
People source mescenchymal stem cell factor opened and preparation method thereof, collects and merges P1-P5 for cell growth to degree of converging 75-85%
When supernatant, and supernatant is filtered and ultrafiltration obtains cell factor concentrate, the method fails to provide effective
Inducing culture, inducing cytokine secretion process is not efficient enough, the mescenchymal stem cell factor negligible amounts for obtaining, and should
Method needs to collect more for cell culture medium, and efficiency is low, and technique is discontinuous, easily causes sample to be polluted by unnecessary, and
Withhold the methods of collection to prepare cell factor, product homogeneity is relatively difficult to ensure card, isolates and purifies process and does not have scale, it is more difficult to suitable more
Should produce in enormous quantities.
The content of the invention
In order to solve the above problems, the invention provides a kind of mescenchymal stem cell factor large-scale producing method,
Erecting and improving sample collection specification, the preparation technology for setting up mass cell culture and secretion inducing cell factor is designed a set of
High-efficiency and continuous separation purifying technique, product can be preserved for a long time through vacuum freeze drying.
To achieve the above object, concrete technical scheme of the present invention is as follows：
A kind of mescenchymal stem cell factor large-scale producing method, comprises the following steps：
1st, the in vitro culture of mescenchymal stem cell：Healthy mescenchymal stem cell is chosen, through primary culture in vitro, Secondary Culture
To 4 generations, 4 generation cell dissociations are counted, going to cell factory carries out amplification cultivation；
2nd, the secretion inducing of the mescenchymal stem cell factor：When the cell of amplification cultivation it is long to degree of converging 70~85% when, abandon
Fall former culture medium, addition inducing culture to cell factory continues to cultivate, and collects inducing culture；
3rd, the mescenchymal stem cell factor is isolated and purified：The inducing culture of collection is followed by hollow fiber filter membrane
Ring is filtered 5-8 times, collects the first thick pure liquid, and by the first thick pure liquid pump to cross-flow ultrafiltration film bag, circulating filtration to first is slightly pure
Liquid volume concentration is the 1/30-1/60 of original volume, then obtains the to adding PBS to mend to original volume in the thick pure liquid of concentration
Two thick pure liquid, continue pump to cross-flow ultrafiltration film packet loop to filter to the second thick neat liquid product concentration is the 1/40-1/ of original volume
After 60, the 3rd thick pure liquid is obtained to adding physiological saline to mend to original volume in the second thick pure liquid of concentration, continuation ultrafiltration is to the 3rd thick
Neat liquid product concentration is the 1/30-1/50 of original volume, collects concentration filtrate and obtains mescenchymal stem cell factor concentrate；
Wherein, the inducing culture is 30-80 by volume ratio:20-60:The DMEM of 0.2-1.5, PBS and L-
Mixed culture medium and the derivant composition of the alanyl-L-glutamine aqueous solution, the derivant include the component of following concentration：
The D-Glucose of HEPES, 1-2g/100ml of 10-20mmol/L and the 40-70 μm of L- vitamin C of ol/L, wherein DMEM's is dense
It is 1500-3000mg/L to spend, and the concentration of PBS is 0.01-0.03M, the concentration of the Ala-Gln aqueous solution
It is 150-300mM.
Mescenchymal stem cell factor large-scale producing method provided by the present invention, is trained on a large scale using single samples sources
Support, fidelity batch products homogeneity, the secretion inducing stage of the mescenchymal stem cell factor adds derivant using basal medium
Inducing culture is configured to, a large amount of secretions of stimulating cytokine, formula rationally effectively, keeps the motility rate high of NSC same
Shi great Liang secrete cytokines, inducing culture is substantially free of high molecular weight protein composition, is easy to late-stage products to isolate and purify；Between fill
The stage that isolates and purifies of matter stem cell factor replaces buffer solution step comprising two steps, and first step PBS is effectively prevented carefully
Intracellular cytokine purge process is inactivated；Second uses physiological saline, it is to avoid PBS is in freeze-drying process because self character is produced
PH drastically change and cause protein inactivation, whole preparation process conditional stability is rationally, suitable for mass production.
Further, the pyridoxol salt of thioglycerol and 0.8-1.3mmol/L of the derivant also including 5-7mg/ml
Hydrochlorate.
Further preferably, when the cell of amplification cultivation it is long to degree of converging 70~85% when, discard former culture medium, addition is former
The inducing culture of culture medium half volume continues to cultivate 72h to cell factory；
Wherein, condition of culture is 37 DEG C, 5%CO in 0-24h2, 5%O2, condition of culture is in 24-48h：37 DEG C, 5%
CO2, 20%O2；
Condition of culture is in 48-72h：37 DEG C, 5%CO2, 5%O2；Inducing culture is measured in collecting half after 48h, and to thin
Equivalent inducing culture is added in born of the same parents factory；Whole inducing cultures are collected after 72h.
Induction period alternately improves culture environment and releases a part of inducing culture, adds fresh inducing culture, from
And improve cell growth environment, and Product inhibiton is released, make the further secrete cytokines of cell.
Further, the preparation method also freeze-drying step including the mescenchymal stem cell factor, mesenchyma is done
Cell factor concentrate normal saline dilution obtains mescenchymal stem cell factor normal saline dilution liquid, adds freeze drying protectant
It is well mixed, -80 DEG C of freezings after filtration sterilization, and be vacuum dried, the mescenchymal stem cell factor freeze-dried powder for obtaining, -20
DEG C preserve.
Further, the freeze drying protectant include 250-400mmol/L trehaloses, 180-220mmol/L sorbierites and
The human serum albumin of 0.8-1.3g/L；Further, the freeze drying protectant also includes：The diaminourea second of 30-50mmol/L
The progesterone of alkane polyacrylamide and 12-36mmol/L, the mescenchymal stem cell factor is effectively protected in freeze-drying process, it is ensured that
The high-quality of freeze-dried powder.
Preferably, the in vitro culture of mescenchymal stem cell comprises the following steps：
1st, in aseptic condition obtains the tissue block containing mescenchymal stem cell loaded on blake bottle, mescenchymal stem cell is added
Serum free medium, is placed in 37 DEG C, 5%CO2Cultivated in incubator, keep blake bottle absolute rest culture 5 days, thereafter every 3 days
Change liquid；
2nd, whne cell is long healed to 75-85% when, abandon old culture medium, in acellular culture face add brine two
It is secondary, 0.25% pancreatin homogeneous immersion cell attachment face is subsequently adding, 3-5min is incubated at room temperature, trained after serum-free is added after cell rounding
Base is supported, cell attachment face is quickly shaken and blow and beat, cell suspension is obtained, supernatant is abandoned in centrifugation, and precipitation plus physiological saline are washed again
And cell precipitation is centrifuged to obtain, the cell precipitation is resuspended with serum free medium, cell sieve filtering, counts and think 1-2 × 104
Individual/ml cell concentrations paving bottle, puts 37 DEG C, 5%CO2Secondary Culture in incubator；
3rd, 4 generation cells of the mescenchymal stem cell factor are taken, digestion is counted and presses 1-2 × 104/ ml density is inoculated into cell factory
In, while blake bottle is inoculated in culture by equal densities, with 37 DEG C, 5%CO2, 20%O2Condition is cultivated.
Using well-fed culture medium, favourable condition of culture, rapid, high volume amplification of mesenchymal stem cells, using hypoxemia
Environment, simulates mescenchymal stem cell environment in vivo, stimulates a large amount of secrete cytokines.
Preferably, the tissue containing mescenchymal stem cell is people's umbilical cord, and acquisition method comprises the following steps：
1st, umbilical cord source pregnant woman is determined in advance, familial inheritance medical history is investigated, and taking out female blood carries out Viral diagnosis, and record health is adjusted
Table look-up；
2nd, neonate's birth gathers umbilical cord on the spot, is stored in accumulating liquid, holding time<12h；
3rd, by umbilical cord sterilization, cleaning, it is cut into small pieces, longitudinally tears, cuts open from 1 radicular vein blood vessel and 2 radicular arteries blood vessels, removal
Amnion, tears and takes huatong plastic；
4th, huatong plastic is washed, is fully shredded to 1mm3-3mm3Size, obtains final product and contains mescenchymal stem cell tissue block.
Above method Erecting and improving sample collection specification, is conducive to the stability of mass cell culture sample quality.
On the other hand, the preparation facilities needed for the present invention provides a kind of mescenchymal stem cell factor large-scale producing method,
Including the cell factory by pipeline successively airtight connection, inducing culture storage tank, hollow-fibre membrane microstrainer, thick pure liquid storage
Tank, cross-flow ultrafiltration film ultrafilter and Biohazard Safety Equipment, the thick pure liquid storage tank are also connected with PBS storage tank by pipeline
With physiological saline storage tank；First circulation pipeline is additionally provided between the hollow-fibre membrane microstrainer and the inducing culture storage tank,
The cross-flow ultrafiltration film ultrafilter and the thick pure liquid storage tank part are additionally provided with second circulation pipeline, the first circulation pipeline,
Second circulation pipeline, between cell factory and inducing culture storage tank, between hollow-fibre membrane microstrainer and thick pure liquid storage tank,
Between PBS storage tank or physiological saline storage tank and thick pure liquid storage tank and cross-flow ultrafiltration film ultrafilter and Biohazard Safety Equipment it
Between pipeline on be equipped with valve, between the hollow-fibre membrane microstrainer and the inducing culture storage tank and cross-flow ultrafiltration
Aseptic pump is also equipped with pipeline between film ultrafilter and the thick pure liquid storage tank part.There is provided the closing of a set of high-efficiency and continuous
Device is isolated and purified, whole purification system airtight connection, continuous operation is greatly improved and isolates and purifies efficiency.
Preferably, the aseptic pump is aseptic peristaltic pump, and the molecular cut off of the cross-flow ultrafiltration film ultrafilter is
5kD。
Further, the inducing culture storage tank, on thick pure liquid storage tank, PBS storage tank and physiological saline storage tank
Air cleaner is equipped with, the entrance of microbes in air or bacterium is effectively prevented.
The beneficial effects of the present invention are:
1st, there is provided systematic sample collection, cell culture, cytokine induction secretion and cell factor isolate and purify work
Skill, single samples sources large-scale culture, maximum possible realizes product quality stability, homogeneity and reappearance, it is adaptable to work
Industry is produced；
2nd, culture is divided into two stages, and the first stage is expanded using nutritional sufficiency culture medium, favourable condition of culture rapid, high volume
Increase cell；Second stage uses low-oxygen environment using basal medium addition derivant configuration inducing culture, is filled between simulation
Matter stem cell environment in vivo, stimulates a large amount of secrete cytokines；
3rd, purge process replaces buffer solution step comprising two steps, and first step PBS effectively prevents cell factor pure
Change process is inactivated；Second uses physiological saline, it is to avoid PBS in freeze-drying process because self character produce PH drastically
Change causes protein inactivation.
4th, inducing culture is substantially free of high molecular weight protein composition, is easy to late-stage products to isolate and purify.Induction period replaces
Improve culture environment and release a part of inducing culture, add fresh inducing culture, so as to improve cell growth environment, solve
Except Product inhibiton, make the further secrete cytokines of cell；
5th, purifying concentrate determines 4 kinds and represents factor concentration using ELISA method, then adjusts concentrate cell factor dense
Degree, finally adding protective agent carries out vacuum freeze drying, vacuum gland, it is ensured that the homogeneity between final products batch.Favorably
In the follow-up application in terms of beauty, preclinical study and medical treatment of product.
Preparation facilities needed for the 6th, a kind of mescenchymal stem cell factor large-scale producing method of holonomic system is provided, the dress
Put and realize the continuous operation that the mescenchymal stem cell factor is prepared on a large scale, greatly improve purification efficiency, and can effectively prevent
Microbes in air enters；The aseptic optional peristaltic pump of pump, is easier to ensure sterile working
Specific embodiment
With reference to embodiment, the present invention is further described；Following embodiments are illustrative, be not it is limited,
Protection scope of the present invention can not be limited with following embodiments；Equipment used in the present invention, unless otherwise required, is
Conventional equipment in the art；Method used in the present invention, unless otherwise required, is conventional method in the art.
Embodiment 1
A kind of mescenchymal stem cell factor large-scale producing method, comprises the following steps：
1st, choose mescenchymal stem cell, through primary culture in vitro, Secondary Culture to 4 generations after, by 4 generation cell dissociations count,
Going to cell factory carries out amplification cultivation；
2nd, when the cell of amplification cultivation it is long to degree of converging 70~85% when, discard former culture medium, addition inducing culture is extremely
Cell factory, continues to cultivate, and collects inducing culture；
3rd, the inducing culture of collection is circulated filtering 5 times by hollow fiber filter membrane, collects the first thick pure liquid, will
To cross-flow ultrafiltration film bag, circulating filtration to the first thick neat liquid product concentration is the 1/50 of original volume to first thick pure liquid pump, then
The second thick pure liquid is obtained, continues pump to cross-flow ultrafiltration film Bao Xun to adding PBS to mend to original volume in the thick pure liquid of concentration
Ring is filtered to the second thick neat liquid product concentration as after the 1/50 of original volume, to added in the thick pure liquid of the second concentration physiological saline mend to
Original volume obtains the 3rd thick pure liquid, and it is the 1/40 of original volume to continue ultrafiltration to the 3rd thick neat liquid product concentration, collects concentration filtrate
Obtain mescenchymal stem cell factor concentrate；
Wherein, the inducing culture is 50 by volume ratio by culture medium:49:1 DMEM, PBS and the ammonia of L- third
Mixed culture medium and the derivant composition of acyl-Glu aqueous solution, the derivant include the component of following concentration：
The D-Glucose of HEPES, 1.3g/100ml of 15mmol/L and 52 μm of L- vitamin Cs of ol/L, the former culture medium are conventional
Serum free medium, wherein DMEM concentration are 2500mg/L, and the concentration of PBS cushioning liquid is 0.02M, L- alanyls-
The concentration of the Glu aqueous solution is 200mM.
Embodiment 2
A kind of mescenchymal stem cell factor large-scale producing method, comprises the following steps：
1st, choose mescenchymal stem cell, through primary culture in vitro, Secondary Culture to 4 generations after, by 4 generation cell dissociations count,
Going to cell factory carries out amplification cultivation；
2nd, when the cell of amplification cultivation it is long to degree of converging 70~85% when, discard former culture medium, addition inducing culture is extremely
Cell factory, continues to cultivate, and collects inducing culture；
3rd, the inducing culture of collection is circulated filtering 8 times by hollow fiber filter membrane, collects the first thick pure liquid, will
To cross-flow ultrafiltration film bag, circulating filtration to the first thick neat liquid product concentration is the 1/30 of original volume to first thick pure liquid pump, then
The second thick pure liquid is obtained, continues pump to cross-flow ultrafiltration film Bao Xun to adding PBS to mend to original volume in the thick pure liquid of concentration
Ring is filtered to the second thick neat liquid product concentration as after the 1/40 of original volume, to added in the thick pure liquid of the second concentration physiological saline mend to
Original volume obtains the 3rd thick pure liquid, and it is the 1/30 of original volume to continue ultrafiltration to the 3rd thick neat liquid product concentration, collects concentration filtrate
Obtain mescenchymal stem cell factor concentrate；
Wherein, the inducing culture is 30 by volume ratio by culture medium:68.5:1.5 DMEM, PBS and L-
Mixed culture medium and the derivant composition of the alanyl-L-glutamine aqueous solution, the derivant include the component of following concentration：
The D-Glucose of HEPES, 1g/100ml of 10mmol/L and 40 μm of L- vitamin Cs of ol/L, the thioglycerol of 5mg/ml and
The pyridoxine hydrochloride of 0.8mmol/L, wherein DMEM concentration are 1500mg/L, and the concentration of PBS cushioning liquid is
0.01M, the concentration of the Ala-Gln aqueous solution is 300mM..
Embodiment 3
A kind of mescenchymal stem cell factor large-scale producing method, comprises the following steps：
1st, choose mescenchymal stem cell, through primary culture in vitro, Secondary Culture to 4 generations after, by 4 generation cell dissociations count,
Going to cell factory carries out amplification cultivation；
2nd, when cell it is long to degree of converging 70~85% when, discard former culture medium, and add according to former culture medium half volume
Inducing culture, continuing culture adjusting parameter is:37 DEG C, 5%CO2, 5%O2, culture parameters are adjusted after 24h is：37 DEG C, 5%
CO2, 20%O2, half amount inducing culture is collected after 48h, and to the fresh inducing culture of equivalent is added in cell factory, adjust ginseng
Number is 37 DEG C, 5%CO2, 5%O2, continue to cultivate；Whole inducing cultures are collected after 72h；
3rd, the inducing culture of collection is circulated filtering 7 times by the hollow fiber filter membrane of 0.1um, collects first thick
Pure liquid, by the first thick pure liquid pump to cross-flow ultrafiltration film bag, circulating filtration to the first thick neat liquid product concentration is the 1/ of original volume
60, then mended to original volume to addition PBS in the thick pure liquid of concentration and obtain the second thick pure liquid, continue pump to cross-flow ultrafiltration
Film packet loop is filtered to the second thick neat liquid product concentration as after the 1/60 of original volume, to adding physiology salt in the thick pure liquid of the second concentration
Water is mended to original volume and obtains the 3rd thick pure liquid, and it is the 1/50 of original volume to continue ultrafiltration to the 3rd thick neat liquid product concentration, is collected dense
Contracting filtrate obtains mescenchymal stem cell factor concentrate
Wherein, the inducing culture is 80 by volume ratio by culture medium:19.8:0.2 DMEM, PBS and L-
Mixed culture medium and the derivant composition of the alanyl-L-glutamine aqueous solution, the derivant include the component of following concentration：
The D-Glucose of HEPES, 2g/100ml of 20mmol/L and 70 μm of L- vitamin Cs of ol/L, the thioglycerol of 7mg/ml and
The pyridoxine hydrochloride of 1.3mmol/L, wherein DMEM concentration are 3000mg/L, and the concentration of PBS cushioning liquid is
0.03M, the concentration of the Ala-Gln aqueous solution is 300mM.
Embodiment 4
A kind of mescenchymal stem cell factor large-scale producing method, comprises the following steps：
1st, choose mescenchymal stem cell, through primary culture in vitro, Secondary Culture to 4 generations after, by 4 generation cell dissociations count,
Going to cell factory carries out amplification cultivation；
2nd, when cell it is long to degree of converging 70~85% when, discard former culture medium, and add according to former culture medium half volume
Inducing culture, continuing culture adjusting parameter is:37 DEG C, 5%CO2, 5%O2, culture parameters are adjusted after 24h is：37 DEG C, 5%
CO2, 20%O2, half amount inducing culture is collected after 48h, and to the fresh inducing culture of equivalent is added in cell factory, adjust ginseng
Number is 37 DEG C, 5%CO2, 5%O2, continue to cultivate；Whole inducing cultures are collected after 72h；
3rd, the inducing culture of collection is circulated filtering 6 times by the hollow fiber filter membrane of 0.1um, collects first thick
Pure liquid, by the first thick pure liquid pump to cross-flow ultrafiltration film bag, circulating filtration to the first thick neat liquid product concentration is the 1/ of original volume
50, then mended to original volume to addition PBS in the thick pure liquid of concentration and obtain the second thick pure liquid, continue pump to cross-flow ultrafiltration
Film packet loop is filtered to the second thick neat liquid product concentration as after the 1/50 of original volume, to adding physiology salt in the thick pure liquid of the second concentration
Water is mended to original volume and obtains the 3rd thick pure liquid, and it is the 1/50 of original volume to continue ultrafiltration to the 3rd thick neat liquid product concentration, is collected dense
Contracting filtrate obtains mescenchymal stem cell factor concentrate；
4th, mescenchymal stem cell factor concentrate is collected to suitable centrifuge tube, sampling ELISA method determine VEGF, BDNF,
GDNF, bFGF concentration, refined solution is obtained with normal saline dilution to debita spissitudo, then refined solution is suctioned out with syringe, 0.22 μ
M membrane filtrations are degerming, be dispensed into 2ml it is aseptic, without thermal source cillin bottle, often pipe 1ml, -80 DEG C of quick-frozen 4h of refrigerator are placed in by cillin bottle；
Cillin bottle is placed in gland-type vacuum freeze drier, floating head is covered, starts freeze drier, vacuum 1.05mbar is set,
Drying time 24h；Drying terminates, vacuum gland, and cillin bottle takes out -20 DEG C of preservations.
Wherein, the inducing culture is 50 by volume ratio by culture medium:49:1 DMEM, PBS and the ammonia of L- third
Mixed culture medium and the derivant composition of acyl-Glu aqueous solution, the derivant include the component of following concentration：
The D-Glucose of HEPES, 2g/100ml of 10mmol/L and 50 μm of L- vitamin Cs of ol/L.
Freeze drying protectant includes：The human serum albumin of 250mmol/L trehaloses, 180mmol/L sorbierites and 0.8g/L.
Embodiment 5
A kind of mescenchymal stem cell factor large-scale producing method, is that freeze drying protectant includes with the difference of embodiment 4：
The human serum albumin of 400mmol/L trehaloses, 220mmol/L sorbierites and 1.3g/L, the diaminoethanes poly- third of 50mmol/L
The progesterone of acrylamide and 36mmol/L.
Embodiment 6
A kind of mescenchymal stem cell factor large-scale producing method, comprises the following steps：
1st, the in vitro culture of mescenchymal stem cell：
1) umbilical cord source pregnant woman is determined in advance, familial inheritance medical history is investigated, and taking out female blood carries out Viral diagnosis, and record health is adjusted
Table look-up；
2) neonate's birth gathers umbilical cord on the spot, is stored in accumulating liquid, holding time<12h；
3) by umbilical cord sterilization, cleaning, it is cut into small pieces, longitudinally tears, cuts open from 1 radicular vein blood vessel and 2 radicular arteries blood vessels, removal
Amnion, tears and takes huatong plastic；
4) huatong plastic is washed, is fully shredded to 1mm3-3mm3Size, obtains final product and contains mescenchymal stem cell tissue block；
5) by tissue block and loaded in blake bottle, mesenchymal stem cell serum-free culture medium is added, is placed in 37 DEG C, 5%CO2
Cultivated in incubator, keep blake bottle absolute rest culture 5 days, change liquid within every 3 days thereafter；
6) whne cell it is long to 80% healing when, abandon old culture medium, in acellular culture face add brine twice,
It is subsequently adding 0.25% pancreatin homogeneous immersion cell attachment face, is incubated at room temperature 3-5min, after after cell rounding plus free serum culture
Base, quickly shakes and blows and beats cell attachment face, obtains cell suspension, and supernatant is abandoned in centrifugation, and precipitation plus physiological saline are washed simultaneously again
Cell precipitation is centrifuged to obtain, the cell precipitation is resuspended with serum free medium, cell sieve filtering counts and think 1-2 × 104
Individual/ml cell concentrations paving bottle, puts 37 DEG C, 5%CO2Secondary Culture in incubator；
7) 4 generation cells of the mescenchymal stem cell factor are taken, digestion is counted and presses 1-2 × 104/ ml density is inoculated into cell factory
In, while blake bottle is inoculated in culture by equal densities, with 37 DEG C, 5%CO2, 20%O2Condition is cultivated.
2nd, the secretion inducing of the mescenchymal stem cell factor：When cell it is long to degree of converging 70~85% when, discard former culture medium,
And inducing culture is added according to former culture medium half volume, continuing culture adjusting parameter is:37 DEG C, 5%CO2, 5%O2, 24h
Adjustment culture parameters are afterwards：37 DEG C, 5%CO2, 20%O2, half amount inducing culture is collected after 48h, and add in cell factory
The fresh inducing culture of equivalent, adjusting parameter is 37 DEG C, 5%CO2, 5%O2, continue to cultivate；Whole Fiber differentiations are collected after 72h
Base；
3rd, the mescenchymal stem cell factor is isolated and purified：The inducing culture that will be collected is filtered by the doughnut of 0.1um
Film is circulated filtering 6 times, collects the first thick pure liquid, by the first thick pure liquid pump to cross-flow ultrafiltration film bag, circulating filtration to the
It is the 1/50 of original volume that one thick neat liquid accumulates concentration, is then mended to original volume to addition PBS in the thick pure liquid of concentration and obtained
Second thick pure liquid, continue pump to cross-flow ultrafiltration film packet loop to filter to the second thick neat liquid product concentration is the 1/50 of original volume
Afterwards, mended to original volume to addition physiological saline in the thick pure liquid of the second concentration and obtain the 3rd thick pure liquid, continuation ultrafiltration to the 3rd is slightly pure
Liquid volume concentration is the 1/50 of original volume, collects concentration filtrate and obtains mescenchymal stem cell factor concentrate；
4th, the freeze-drying of the mescenchymal stem cell factor, by mescenchymal stem cell factor concentrate normal saline dilution,
Addition freeze drying protectant is well mixed, -80 DEG C of freeze-dryings after filtration sterilization, the mescenchymal stem cell factor freeze-dried powder for obtaining, -
20 DEG C of preservations；
Wherein, the inducing culture is 50 by volume ratio by culture medium:49:1 DMEM, PBS and the ammonia of L- third
Mixed culture medium and the derivant composition of acyl-Glu aqueous solution, the derivant include the component of following concentration：
The D-Glucose of HEPES, 1.5g/100ml of 10mmol/L and 55 μm of L- vitamin Cs of ol/L.
Freeze drying protectant includes：The human serum albumin of 250mmol/L trehaloses, 180mmol/L sorbierites and 0.8g/L,
The diaminoethanes polyacrylamide and the progesterone of 12mmol/L of 30mmol/L.
Embodiment 7
A kind of preparation facilities needed for mescenchymal stem cell factor large-scale producing method, including it is closed successively by pipeline
The cell factory of connection, inducing culture storage tank, slightly hollow-fibre membrane microstrainer, pure liquid storage tank and cross-flow ultrafiltration film ultrafiltration
Device, the thick pure liquid storage tank is also connected with PBS storage tank and physiological saline storage tank by pipeline；The hollow-fibre membrane is micro-
First circulation pipeline is additionally provided between filter and the inducing culture storage tank, the cross-flow ultrafiltration film ultrafilter is thick pure with described
Second circulation pipeline, the first circulation pipeline, second circulation pipeline, cell factory and inducing culture are additionally provided between liquid storage tank
Between storage tank, between hollow-fibre membrane microstrainer and thick pure liquid storage tank, PBS storage tank or physiological saline storage tank and thick pure liquid
Valve, the hollow-fibre membrane are equipped with pipeline between storage tank and between cross-flow ultrafiltration film ultrafilter and Biohazard Safety Equipment
Pipe between microstrainer and the inducing culture storage tank and between cross-flow ultrafiltration film ultrafilter and the thick pure liquid storage tank
Aseptic pump is also equipped with road, it is preferable that the aseptic pump is aseptic peristaltic pump, the retention of the cross-flow ultrafiltration film ultrafilter
Molecular weight is 5kD.
Embodiment 8
A kind of preparation facilities needed for mescenchymal stem cell factor large-scale producing method, the difference with embodiment 7 is,
Air cleaner is equipped with inducing culture storage tank, thick pure liquid storage tank, PBS storage tank and physiological saline storage tank.
Comparative examples 1
A kind of mescenchymal stem cell factor large-scale producing method, the difference with embodiment 1 is, inducing culture with it is former
Culture medium is identical, and the former culture medium is conventional serum free medium.
Comparative examples 2
A kind of mescenchymal stem cell factor large-scale producing method, the difference with embodiment 1 is, in inducing culture,
Concentration of the component and each component that derivant includes in inducing culture is as follows：HEPES, 1g/100ml's of 10mmol/L is sweet
Dew sugar, the vitamin E of 50 μm of ol/L.
Comparative examples 3
A kind of mescenchymal stem cell factor large-scale producing method, the difference with embodiment 1 is, the inducing culture
Middle culture medium is 50 by volume ratio:50 DMEM and PBS is mixed.
Comparative examples 4
A kind of mescenchymal stem cell factor large-scale producing method, the difference with embodiment 1 is, in inducing culture,
Concentration of the component and each component that derivant includes in inducing culture is as follows：The D- of HEPES, 1g/100ml of 10mmol/L
The thioglycerol of glucose, the L- vitamin Cs 5mg/ml of 50 μm of ol/L.
Comparative examples 5
A kind of mescenchymal stem cell factor large-scale producing method, the difference with embodiment 4 is, the freeze drying protectant
To contain following component in every 100ml：Ascorbic acid (VC) 0.5g, human albumin 0.5ml, dextran 3.5g, trehalose
1.5g, glycine 0.04g, arginine 0.17g, amion acetic acid 2g, sodium citrate 0.26g, tert-butyl alcohol 15ml.
Experiment 1：The assay of cell factor in mescenchymal stem cell factor concentrate
Take the people source mesenchyma disclosed in equivalent embodiment 1-3, reference examples 1-4 and Chinese patent application CN105543313A
Mescenchymal stem cell factor concentrate prepared by stem cell factor preparation method, using ELISA method determine VEGF, BDNF,
GDNF, bFGF concentration, the results are shown in Table 1.
The concentration of each cell factor of table 1
| Group | VEGF | BDNF | GDNF | bFGF |
| Embodiment 1 | 457ng/ml | 269ng/ml | 206ng/ml | 353ng/ml |
| Embodiment 2 | 537ng/ml | 321ng/ml | 257ng/ml | 398ng/ml |
| Embodiment 3 | 602ng/ml | 364ng/ml | 283ng/ml | 435ng/ml |
| Reference examples 1 | 211ng/ml | 137ng/ml | 89ng/ml | 159ng/ml |
| Reference examples 2 | 397ng/ml | 221ng/ml | 153ng/ml | 301ng/ml |
| Reference examples 3 | 335ng/ml | 158ng/ml | 183ng/ml | 226ng/ml |
| Reference examples 4 | 462ng/ml | 285ng/ml | 213ng/ml | 363ng/ml |
| CN105543313A | 385ng/ml | 214ng/ml | 164ng/ml | 297ng/ml |
As can be seen from Table 1, compared with reference examples 1-3, high degree improves the concentration of each cell factor to embodiment 1,
There is good inducing action to mescenchymal stem cell secrete cytokines, comparative examples 2 are with the difference of embodiment 1,
D-Glucose is replaced with into its isomer mannose, L- vitamin Cs are replaced with into vitamin E of the same clan；Comparative examples 3
It is without the Ala-Gln aqueous solution in mixed culture medium in inducing culture with the difference of embodiment 1；Examination
Test result and understand that the inducing culture that above-mentioned change is constituted can not preferably play inducing mesenchymal stem cell secretory cell
The effect of the factor, each component is mutually cooperateed with, indispensable and irreplaceable；Embodiment 2 further optimizes relative to embodiment 1
The component of inducing culture, has experimental result to understand the cytokine concentrations of embodiment 2 higher than embodiment 1, reference examples 4 with it is real
The difference for applying example 2 is different inducing culture component, and as seen from the experiment, the mescenchymal stem cell factor of embodiment 2 is lured
Lead secretion effect and be much better than reference examples 4；And embodiment 3 optimizes the condition of cell culture on the basis of embodiment 2, more enter
One step improves the amount of cell secretion of cytokines；It is dry thin mesenchyma to be prepared with the method for Chinese patent application CN105543313A
Intracellular cytokine quantity compares, and the cell factor that mescenchymal stem cell factor preparation method provided by the present invention is obtained is more, effect
Rate is high, and method is simple.
Experiment 2：Mescenchymal stem cell factor freeze-dried powder is checked
The mescenchymal stem cell factor freeze-dried powder of Example 4, embodiment 5 and the gained of comparative examples 2, puts in -10 DEG C
Put 18 months, proterties, residual moisture and the bFGF activity that freeze-dried powder was observed when 6 months, 12 months and 18 months are carried out
Detection, testing result is shown in Table 2.
The test group of table 2 and the neural stem cell differentiating result of control group
Understood through test result analysis, freeze drying protectant provided by the present invention, the mesenchyma that can form stabilization is dry thin
Intracellular cytokine freeze-dried powder, make product quality stabilization, it is easy to preserve, can be placed under the conditions of -10 DEG C more than 12 months it is unchanged, it is aqueous
Amount is below 1.33%, and the freeze drying protectant that wherein embodiment 5 is provided is better compared with embodiment 4；Embodiment 1 and reference examples
2 compared to better, and composition is simple, with low cost.
Claims (10)
1. a kind of mescenchymal stem cell factor large-scale producing method, it is characterised in that comprise the following steps：
(1) in vitro culture of mescenchymal stem cell：Mescenchymal stem cell is chosen, through primary culture in vitro, Secondary Culture to 4 generations
Afterwards, 4 generation cell dissociations are counted, going to cell factory carries out amplification cultivation；
(2) secretion inducing of the mescenchymal stem cell factor：When the cell of amplification cultivation it is long to degree of converging 70~85% when, discard original
Culture medium, addition inducing culture to cell factory continues to cultivate, and collects inducing culture；
(3) the mescenchymal stem cell factor is isolated and purified：The inducing culture of collection is circulated by hollow fiber filter membrane
Filtering 5-8 times, collects the first thick pure liquid, by the first thick pure liquid pump to cross-flow ultrafiltration film bag, circulating filtration to the first thick pure liquid
Volume concentration is the 1/30-1/60 of original volume, is then mended to original volume to addition PBS in the thick pure liquid of concentration and obtains second
Thick pure liquid, continue pump to cross-flow ultrafiltration film packet loop to filter to the second thick neat liquid product concentration is the 1/40-1/60 of original volume
Afterwards, mended to original volume to addition physiological saline in the thick pure liquid of the second concentration and obtain the 3rd thick pure liquid, continuation ultrafiltration to the 3rd is slightly pure
Liquid volume concentration is the 1/30-1/50 of original volume, collects concentration filtrate and obtains mescenchymal stem cell factor concentrate；
Wherein, the inducing culture is formed by derivant and mixed culture medium configuration, and the mixed culture medium is by volume ratio
30-80:19.8-68.5:The PBS and 150-300mM of DMEM, 0.01-0.03M of the 1500-3000mg/L of 0.2-1.5
Ala-Gln aqueous solution composition, component that the derivant includes and each component are in inducing culture
Concentration is as follows：The D-Glucose of HEPES, 1-2g/100ml of 10-20mmol/L and the 40-70 μm of L- vitamin C of ol/L.
2. mescenchymal stem cell factor large-scale producing method as claimed in claim 1, it is characterised in that the derivant is also
Component including following concentration：The thioglycerol of 5-7mg/ml and the pyridoxine hydrochloride of 0.8-1.3mmol/L.
3. mescenchymal stem cell factor large-scale producing method as claimed in claim 1, it is characterised in that mescenchymal stem cell
The specific method of factor secretion inducing is as follows：
When the cell of amplification cultivation it is long to degree of converging 70~85% when, discard former culture medium, the former culture medium half volume of addition
Inducing culture continues to cultivate 72h to cell factory；
Wherein, condition of culture is 37 DEG C, 5%CO in 0-24h2, 5%O2, condition of culture is in 24-48h：37 DEG C, 5%CO2、
20%O2；
Condition of culture is in 48-72h：37 DEG C, 5%CO2, 5%O2；Inducing culture is measured in collecting half after 48h, and to cell work
Equivalent inducing culture is added in factory；Whole inducing cultures are collected after 72h.
4. the large-scale producing method of the mescenchymal stem cell factor as claimed in claim 1, it is characterised in that the preparation side
The method also freeze-drying step including the mescenchymal stem cell factor, by mescenchymal stem cell factor concentrate normal saline dilution
Obtain mescenchymal stem cell factor normal saline dilution liquid, addition freeze drying protectant is well mixed, after filtration sterilization -80 DEG C it is cold
Freeze, and be vacuum dried, the mescenchymal stem cell factor freeze-dried powder for obtaining, -20 DEG C of preservations.
5. mescenchymal stem cell factor large-scale producing method as claimed in claim 4, it is characterised in that the frozen-dried protective
Concentration of the component and each component that agent includes in mescenchymal stem cell factor normal saline dilution liquid is as follows：250-400mmol/
The human serum albumin of L trehaloses, 180-220mmol/L sorbierites and 0.8-1.3g/L.
6. mescenchymal stem cell factor large-scale producing method as claimed in claim 5, it is characterised in that the frozen-dried protective
Agent also includes：The diaminoethanes polyacrylamide and the progesterone of 12-36mmol/L of 30-50mmol/L.
7. mescenchymal stem cell factor large-scale producing method as claimed in claim 1, it is characterised in that mescenchymal stem cell
In vitro culture comprise the following steps：
(1) by the tissue block containing mescenchymal stem cell, and loaded in blake bottle, mesenchymal stem cell serum-free culture is added
Base, is placed in 37 DEG C, 5%CO2Cultivated in incubator, keep blake bottle absolute rest culture 5 days, change liquid within every 3 days thereafter；
(2) when the cell degree of converging to 75-85% long, mesenchymal stem cell serum-free culture medium is abandoned, is added in acellular culture face
Enter brine twice, be subsequently adding 0.25% pancreatin homogeneous immersion cell attachment face, be incubated at room temperature 3-5min, treat cell
Add serum free medium after being rounded, quickly shake and blow and beat cell attachment face, obtain cell suspension, supernatant is abandoned in centrifugation, and precipitation adds
Physiological saline washs and is centrifuged and to obtain cell precipitation again, and the cell precipitation is resuspended with serum free medium, cell sieve filtering, meter
Count and think 1-2 × 104Individual/ml cell concentrations paving bottle, puts 37 DEG C, 5%CO2Secondary Culture in incubator；So repeatedly to cell
It was passaged to for 4 generations；
(3) learn from else's experience the 4th generation mescenchymal stem cell cell of Secondary Culture；By 1-2 × 104/ ml density is inoculated in cell factory and carries out
Amplification cultivation, condition of culture is 37 DEG C, 5%CO2, 20%O2Culture.
8. the preparation facilities needed for a kind of mescenchymal stem cell factor large-scale producing method as claimed in claim 1, it is special
Levy and be, cell factory that the preparation facilities includes being sequentially communicated by pipeline, inducing culture storage tank, hollow-fibre membrane are micro-
Filter, thick pure liquid storage tank and cross-flow ultrafiltration film ultrafilter, the thick pure liquid storage tank are also connected with PBS and store up by pipeline
Tank and physiological saline storage tank；First circulation pipe is additionally provided between the hollow-fibre membrane microstrainer and the inducing culture storage tank
Road, second circulation pipeline, the first circulation pipe are additionally provided between the cross-flow ultrafiltration film ultrafilter and the thick pure liquid storage tank
Road, second circulation pipeline, between cell factory and inducing culture storage tank, hollow-fibre membrane microstrainer and thick pure liquid storage tank it
Between, between PBS storage tanks or physiological saline storage tank and thick pure liquid storage tank and cross-flow ultrafiltration film ultrafilter and Biohazard Safety Equipment between
Pipeline on be equipped with valve, between the hollow-fibre membrane microstrainer and the inducing culture storage tank and cross-flow ultrafiltration film
Aseptic pump is also equipped with pipeline between ultrafilter and the thick pure liquid storage tank.
9. preparation facilities as claimed in claim 8, it is characterised in that the aseptic pump is aseptic peristaltic pump, the slipstream
The molecular cut off of milipore filter ultrafilter is 5kD.
10. preparation facilities as claimed in claim 8, it is characterised in that the inducing culture storage tank, thick pure liquid storage tank, PBS
Air cleaner is equipped with buffering liquid storage tank and physiological saline storage tank.
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| CN106754639B (en) | 2020-06-09 |
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